Diethyl alpha phosphonate as an (antimicrobial agent) algaecide

ABSTRACT

Diethyl alpha allyl phosphonate can be used to inhibit and/or prevent growth of undesirable algae, bacteria, fungi, yeast, and other microorganisms. This invention is particularly concerned with the bacteriostatic and bactericidal properties of diethyl alpha allyl phosphonate compounds against species of Proteus, Salmonella, Shigella, Staphylococcus and Xanthomonas.

United States Patent Kerst et a1. Nov. 4, 1975 [5 DHETHYL ALPHA PHOSPHONATE AS AN 3,764,677 10/1973 K616161111. 71/67 (ANTIMICROBIAL AGENT) ALGAECIDE [75] Inventors: af j fi 16 2" Primary Examiner-James 0.. Thomas, Jr.

Attorney, Agent, or Firm-Raymond Fink; H. W. [73] Assignee: The Gates Rubber Company, Oberg, Jr.; Curtis H. Castleman, Jr.

Denver, C010. 1

[22] Filed: Mar. 12, 1973 l [21] Appl. No.: 340,404 57 ABSTRACT Related US. Application Data [62] Division of S61. NO. 84,903, 061. 28, 1970, Pat. NO. DethYl alpha allyl Phosphonatfi F be used to 3,783,161) and/or prevent growth of undeslrable algae, bacteria, fungi, yeast, and other microorganisms. This invention 52 us. 01. 71/67 is particularly concerned with the bacteriostatic and [51] Int. Cl. AOlN 9/36 bactericidal properties Of diethyl alpha allyl phospho- [58] Field of Search 71/86, 67 Hate Compounds against Species Of Proteus, Salmonella, Shigella, Staphylococcus and Xanthomonas. [56] References Cited UNITED STATES PATENTS 10/1973 Kerst et a1. 71/67 11 Claims, N0 Drawings DIETHYL ALPHA PHOSPI-IONATE AS AN (ANTIMICROBIAL AGENT) ALGAECIDE This is a division of application Ser. No. 84,903, filed on Oct. 28, 1970, now issued as U.S. Pat. No. 3,783,160.

BACKGROUND OF THE INVENTION SUMMARY OF THE INVENTION According to the present invention, it has been found that diethyl alpha allyl phosphonate C,H ,o

/P-CH. -CH=CH. C. ,H -,o o

is a very effective antimicrobial agent. The compound diethyl alpha allyl phosphonate is well known and may be prepared according to the methods disclosed by Ford-Moore, A. H. and Williams, J. H., J. Chem. Soc.

1947, 1465. The diethyl alpha allyl phosphonate used to establish the present invention was prepared by two different procedures. The first procedure involved the reaction of substantially equimolar proportions of sodium diethyl phosphite and allyl bromide in ethanol. The reaction took place smoothly in the temperature range of from to 100 C with the preferred reaction conditions being from to 65 C. for about one hour. Following completion of the reaction, the precipitated sodium bromide was removed by filtration and the ethanol was evaporated off under vacuum. The remaining syrup was distilled under vacuum and produced a 65 to 70% yield of diethyl alpha allyl phosphonate.

In an alternative procedure, the diethyl alpha allyl phosphonate compounds of the present invention were prepared by reacting triethyl phosphite with allyl bromide according to the Arbusov method. Good results were obtained by employing substantially equimolar proportions of the reactants. This reaction took place readily at temperatures of from 90 to 100 C. to produce the diethyl alpha allyl phosphonate and a ethyl bromide by-product which was collected by a dry ice condensor. The final mixture was distilled to give an 85 to 90% yield of the diethyl alpha allyl phosphonate.

DESCRIPTION OF THE PREFERRED EMBODIMENTS The following examples illustrate the antimicrobial qualities of diethyl alpha allyl phosphonate and describe how these antimicrobial qualities may be utilized in various phases of agriculture, animal husbandry, pharmacology and water treatment technology.

EXAMPLE I ANTIBACTERIAL AND ANTIYEAST ACTIVITY The in vitro effectiveness of diethyl alpha allyl phosphonate against bacteria and yeast species was established in the following manner. One loopful of each of the investigated bacterial or yeast was transferred from agar slants to 10 ml. of trypticase soy broth and incubated at 37 for 18 hours. At the end of this period, the bacteria or yeast was seeded into the same medium 1.5-27r agar) in which the original inoculum was prepared. The bacteria were then seeded at 1 ml. of inoculum per 250 ml. of medium. which were equivalent to at least 1 X 10 cells/m1. determined by dilution platecount or nephelometer readings. The resultant mixtures were poured into heat-resistant sterile petri dishes at a temperature of 45 C. Analytical filter paper discs 1.2 cm. in diameter were used for the agar diffusion technique. Each disc was saturated with 0.08 ml. of the solubilized diethyl alpha allyl phosphonate compound ug./disc) and placed on the surface of the hard ened agar. The plates were incubated at 37 C. for 18 hours. The activity of the diethyl alpha allyl phosphonate compounds was established by measuring the zone of inhibition in centimeters. Untreated control plates were used as a basis for comparison and these exhibited a profuse growth of bacteria. The results of these tests were as follows:

Zone of Inhibition in Cemimeters SHepIuem'euS lxemulvlle Group B ATCC N0. 9637 Shigella lmvdll ATCC No. 9212 Shigella buvdii ATCC No. 9905 Shigella sonnei MMV 6654 Slligella jlevneri TYPE 6 MM\' 760 V Slligella flevneri TYPE 4 MMV6625 S/zigella dvv'enlerl'ae TYPE 2 MMV 6673 Salmonella panama ATCC No. 7378 Salmunella paralypln' ATCC No. 9281 Salmonella enlerilis ATCC No. 13076 Sullnunella pullarnm ATCC No. 10398 Sallnnnella :lerhy ATCC No. 6960 Salmonella gallinarium ATCC No. 9184 Salmonella lvpliimarimn SR-l 1 Salmonella vpl10.va ATC'C No. 19403 eitreriu gunurl'lmeae ATCC No. 19424 Aeisseria inlraeelluluris ATCC No. Available on Request [\eisserl'a meningilillex ATCC No. 13077 Listeria lnmlurymgenex ATCC No. 15813 lihrin fetus ATCC No. 15296 liln'ln elmlel'ae ATCC No. 14035 Pruleux vnlgnrix -continued Gram positive and Gram negative bacteria Zone of Inhibition in Centimeters ATCC No. 4984 Erwiniu earoiovora 3.6 ATCC No. 495

M vcolmelerium l7lll \'l'l('lllll 2.3 ATCC No. 11314 Myrohaeterimn fortuitum. Debos 2.4 ATCC No. 4243 Myt'olmclerimu avium 1.9 ATCC No. 19421 Myt'olmt'lerium lmvix 2.0 ATCC No. 19274 M \'l()l)(l(ltl'llllll phlei 2.8 ATCC No. 11782. phage host Klelasiellu pueunloniae 2.7 ATCC No. Available on Request Micrat'ot't'ux Ieu'ugena .9 ATCC No. 10875 Mierotmk'us Inelilenxix 3.5

ATCC No. 19399 Micrm-oeeux lysotleiklit'ux 3.7 ATC C No. 4698 ATCC No. Available on Request Baeleroidex pueumoxiules ATCC No. Available on Request BaeIeroiz/ex .verpem' ATCC No. Available on Request Bruce/la almrlus' ATCC No. 4315 Brueella melilensis ATCC No. 19396 Brueella .s'uix ATCC No. 4312 EXAMPLE ll SECONDARY SCREEN ANTIBACTERIAL ACTIVITY To further define the scope of this invention. second ary screening tests using the techniques described in the primary screening tests were also employed against selected bacteria genera at various other concentrations. The results of these secondary screens were as follows:

Compound: Diethyl alpha allyl Concentration (ug/disc) phosphonate MICROORGANISM 53.00 19.80 6.53

Salmonella sp. 3.2 2.6 1.7 ATCC N0. 9120 Salmonella vphimurium mil'aglia 2.9 .0 1.4 SR-l l Salmonella N0. 45 3.6 2.6 1.7h MMU I063 Salmonella B/d 3 l 2.5 1.7h MML' 6674 Salmonella 3 8 2.3 I 5 MMU 6668 Shigellu boy- Iii 4.5 2.7 2 5 ATCC No. 9212 Shigella boyllii 3.6 1.8 1.6 ATCC No. 9905 Shigella flexneri Type 6 4.4 3.0 2.0h MML' 760 Shigella jle.vneri Type 4 3.3 2.7 2 2 MML' 6625 Shigella dysenleriae Type 2 3.7 2.7 2 2 -continued Compound: Diethyl alpha allyl phosphonate MICROORGANISM MMU 6673 .S'higella .\'ounei MMU 6654 PSl'lltlOIHUllllA' aeruginosa ATCC NO. 10145 Psemlomouax aeru t'iuosa ATCC No. 8709 Psemlomonas aeruginoxa ATCC No. 12055 Pseuzlomonax aeru t inosa 28 Pxeudomonaa mallophilia 4.3 G 107 Pseudonmuas K997 2.9 Pxemlomonas K966 3.0 Proleus vulgar-iv 2.8 ATCC No. 9484 Proteus vulgaris 3.4 ATCC No. 8427 Proteus vulgal'ix ATCC No. 4699 Proteusvulgaris- 3.1 ATCC No. 6896 Proteus vulgar-ix ATCC No. 9920 Proteus vulgarix 2.7 2.1 O ATCC No. 6897 Proteus vulgaris ATCC No. 12454 Prmeux mirahilis ATCC No. 9961 Proteus umrgunii (i951 Proteux mirahi/i.\' G9 I 2 Proleux mirahilis K723 Concentration (ug/disc) trh Comments:

11 hazy zone I trace This data indicates that diethyl alpha allyl phosphonate compounds can be used to inhibit many important types of diseases. For example, they can be used against:

Bacteria Disease Produced Staphylococcus aureus SII'('])IU(U('L'HS jaeealix Escherichia eoli Proleus vulgarir PSL'IHIOHHHHLY aeruginosa Major cause of bovine mastitis Bacterial endocarditis Cause of scours and secondary cause of bovine mastitis Major cause of fish disease (trout. pike) Numerous animal and plant in- Elhlllltl cariovora Xaulhomonas p/taxeoli The demonstrated antibacterial activity of diethyl alpha allyl phosphonate compounds against Staphylococcus aureus and Escherichia coli is of particular interest to the field of pharmacology since the disclosed activity indicates that these compounds can be formulated as powders, salves. and ointments for administration in the treatment of burns and bacterially induced inflammations such as abscesses. dermatitis. rashes and the like, particularly in domestic animals.

Although the precise mode of action whereby diethyl alpha allyl phosphonate inhibits bacteria growth is not completely understood, it is believed that the .diethyl alpha allyl phosphonate compounds of this invention may serve as chemical antagonists; that is, as chemicals which compete with enzymes essential to the development of such bacteria. Since enzymes perform their catalytic function by virtue of their affinity for their natural substrate; any compound resembling a substrate in its chemically critical aspect may also have an affinity for the enzymes. If this affinity is great enough, the analog will displace the normal substrate from the enzyme and will prevent the growth reaction from taking place. It is believed that diethyl alpha allyl phosphonate has a chemical affinity for an essential site on one enzyme necessary for bacterial growth and life.

The diethyl alpha allyl'phosphonate formulations of this invention can also contain other therapeutically valued supplements such as local anesthetics, irradiated oils, and other medicinal substances. When used for these or similar purposes, diethyl alpha allyl phosphonate may be incorporated in any therapeutically acceptable carrier such as oils, salvesand ointments, together with adjuvants comprising surface active agents, detergents, dispersing agents, stabilizers and other modifiers which may facilitate the handling and application of the antibacterial material. In the case of the in vitro applications of the compositions of this invention, it is difficult to predict with precision what in all cases will constitute a therapeutic dose even on a weight basis. Variable factors such as type, duration and severity of infection and mode of administration may bedetermining factors for the establishment of therapeutic doses.

Those skilled in the art will recognize that the above data indicates that the scope of this invention should not be limited to any particular disease species or to any particular type of animal or plant life. For example, the noted activity of diethyl alpha allyl phosphonate against Xanthomonas p/zaseoli suggests that this compound will also prove to be of value against such other Xanthomonas species as Xanthomonas translusc'ens, Xanthomonasjuglandis, Xanthomonas resicatoria, Xanthomonas barbareae, Xanthomonas pelargonii, Xanthomonas alfalfae, Xanthomonas vascular-um, et cetera. Xanthomonas species are known to cause diseases of tomatoes, sugar cane, rice, sugar beets, cotton, walnuts, wheat, rye, barley, and beans. Some of the more noteworthy Xanthomonas related diseases are Xanthomonas vesic'atoria (Bacterial Leaf Spot of Tomatoes), Xanthomonas phaseoli (Common Bacterial Blight of Bean), Xanthomonas vasculorum (Gumming Disease of Sugar Cane) and Xanthomonas malvacearum (Bacterial Blight of Cotton).

EXAMPLE III INHIBITION OF XANTHOMONAS VESICATORIA to 8-leaf growth stage, were mounted on a compound sure with an aqueous cell suspension of Xanthomonas yesicatoria containing five percent Carborundum and then immediately placed in an incubation chamber maintained at F. and plus relative humidity. After 40 hours in the incubation chamber, the plants were removed to the greenhouse for further development of the infection. Disease severity was determined by counting the lesions present on the treated leaves. The effectiveness of treatment was determined by direct comparison of the treated plants with the untreated ones. Streptomycin sulfate was used as a reference control antibacterial agent. The results of these tests were as follows:

/z Inhibition at 500 ppm: 54%

"/1 Inhibition at 1000 ppm: 54);

Control Streptomycin Control Concentration ppm EXAMPLE IV ANTIFUNGAL ACTIVITY The antifungal activity of diethyl alpha allyl phosphonate compounds was established by treating Fusarinm 0.\' \sp0runz, Fusarium roseum, R/liZOlJllS nigricans. Rhizopus stolumj'er, Aspergillus niger and Alternaria solum' test fungi in the following manner:

One loopful of each of the tested viable fungi cultures, spores and mycelia was transferred from an agar slant to an 80 ml. portion of the nutrient broth composed of oatmeal agar, Czapeks, Sabouraud and deoinized water to volume. The 80 ml. portion of the fungi and broth was then placed in a sterile shake flask (300 ml.) and the flask was placed on a rotary shaker for 96 to hours at room temperature. At the end of this incubation time period, 10 ml. of the liquid were homogenized and placed into another sterile shake flask (300 ml.) containing 80 ml. of the above nutrient broth and 60 ppm of the inhibitor being evaluated. The flasks were placed on a rotary shaker operating at 240 rpm at room temperature for 3 to 9 days. After this second incubation time, the flasks are taken off and examined for visible fungal growth and mycelia] weights are determined. Untreated controls were used as the basis of comparison and these displayed profuse fungal growths containing species of Fusarium, Rhizopus, Aspergillus and Alternaria. The results of these tests indicated that the diethyl alpha allyl phosphonate compounds of this invention imparted a substantial degree of inhibition of fungal growth at 60 ppm.

EXAMPLE V ANTIFUNGAL AND ANTIYEAST ACTIVITY To further define the antifungal activity of diethyl alpha allyl phosphonate the seeded agar plates were prepared by transferring the cultures from slants washed with saline or phosphate buffer and then transferred to the surface of hardened Sabouraud-Dextrose agar plates. Again, as in the case of Example I, the di ethyl alpha allyl phosphonate was tested by impregnating filter paper discs 1.27 cm. in diameter) with 0.08 ml. of the solubilized diethyl alpha allyl phosphonate compounds I00 ug/disc) and placed on the surface of the hardened agar. The plates were incubated at 30 C. for 18 hours. The activity of diethyl alpha allyl phosphonate was established by measuring the zone of inhibition in centimeters. Untreated control plates were used as a basis for comparison and these exhibited a profuse growth of bacteria. The results of these tests cominued were 215 fOllOWSZ Concentration (ug/disc) MlCROORGANlSM 85.0 28.0 8.0

Zone of Inhibition ATCC No. 10833 Microorganism in Centimeters 'l'ritlmp/rrmn munmgmp/zylcs 4.8 3.5 2.1

. ATCC No. 9129 AXIH'I'JUHILY kk 2.4 'I'riv/mp/rvmn manragr'aplz rlux 5.0 3.9 2.0 ATCC N0. lUU-l ATCC N0. 82l5 Altarnaria .ralani 2.4 'l'ricliopllymn mnsm'unx 6.4 3.8 2 5 ATCC No. 63% ATCC No. 10217 Rliiwpus .rlalaniji'r 2.9 (m- 111 mm haul-01a 3.5 .0 2.6 ATCC No. 10404 ATCC No. 12825 Fasariuni 0.\' \xr mrmn 3.0 Pyl/zimn (HT/ll!IrlllltI/IUX 3 I 2.l 0 UFCC 1122 ATCC No. 12531 Candida (II/Iit'lHIX 3.0 Helm!)illms mrium 01-11110 2.7 2.4 0 SR] 523 ATCC No. 11000 Comments: 11g no growth. 15

EXAMPLE Vl SECONDARY FUNGAL SCREEN EXAMPLE Vll SECONDARY CANDIDA SCREEN A secondary screen using the techmques of Example V produced the following results at the diethyl alpha A secondary of yeast of the genus ,Candlda any] phosphonate Concentrations indicated: produced the following results when the techniques of Examples V and VI were employed at the concentrations indicated: (.oncentratlon (ug/disc) MlCROORGANlSM 85.0 28.0 8.0

. Concentration (ug/disc) Rlmdatmala .11). 2.0 l.6 0 Duke 6 s MlCROORGANlSM 53.0 17.0 RlIi:,upu.\' .rlalunll/i'r 3.0 1.8 0 e a q ATCC NO. 10404 ggldula a/Incam l.7h Monaxtm' purpm'ca 4.5 2.2 0 Cum/Mu "UH-mm 23 L7 ATCC No. 752 U Candida alln'cans 3.0 l.7h P Q ATCC No. H651 IllXflI'IlHH o.\' \'.\'parum (aha/1x0 4.0 .0 0 candid 2'3 g UFCC 1122 Duke 2548 01 P- 3 4 373 cumin/t1 alhit'am .6 1.911 ()b OL' 0 Candida alhicans 2.5 l.9h 5R1 V Mt. Vernon SR1 220 -v l l s 33 It 0 Candida albic'anx 2.9 l.6h .-l.rpr'rglllu.\ \'da\ri 5.3 4.7 2.0 35 ML Vernon SR1 331 9 Candida 5p. 2.: 1.7 Axpcrglllux Illt/ll/(HIS 5 8 tr 0 Mt Vernon ATCC No- 10074 Candida Sp. 2.2 1.7 Axpz'millux flanm' 3.9 2.0 0 ML vgmon 512 ATCCNQ 9643 Candida Sp. 2.5h l.7 Aspargdlux anmlcladam! 3.3 1.7 0 Climb-d, Sp. 2 3 1 7 ATCC No. 1001 40 Mt. Vernon 950 Axpergillux jumigamx 3.2 2.0 0 m- 1 L0 ('epltalux mriinn acronmnimn 3.3 1.9 0 ob- A TCC N 10141 Candida guillivrlnondii 2.7 1.8 Leplialus mriian sp. ng ng 0 0U 0U Candida trupicalix 2.2 1.6 Plmlna IHZIJHHHI'UYOIU 4.5 3.0 2.0 Duke 2847 ATCC N 12569 Candida p.\'1lvdalmpit'ulis' 1.9 tr P!((i[l)lll \ariuli 5.1 4.0 0 Duke 25 3 U Columbia ATCC H14 Candida inlt'rlnedl'a 8 2.5 Mgruspuru .rpliat'nm 3.l 2 5 0 Duke 3 05 CBS ATCC NO- H637 Candida :avhnmidar 3.5 246 :HISil/itl .s'pinasa 3 7 2.1 0 Duke 3 09 ATCC No. 6648 Mm'm' racamuxas 0 Comments: h hazy zone tr trace ATCC No. l2l6 Tltamnidiam ('lvgans 3.5 2.5 tr g 5:23 n m x a l 9 n It will also be recognized by those skilled in the art '(Y 4 (5' 1' A A'l'CC No. 9984 that other protectant, systemic and eradicant procegtpgglq m 1 18 61) 0 dures may provlde detection of other biological activio. Pmkimm "Mum, 6'4 35 ties. Pathogens representative of P/1 conrtcetes, Asw OLJ mycetes, BHSI1IOI7I \([S and the Fungi Imperfeca may v gym? 0 provide indtces of other fungicidal activity. Additional b WI, m pathogens and appropriate host plants may well afford M\' 1919 other opportunities to further define the degree and P/n'aluplmra l'tI'I'llClM'll 5.2 .0 tr f h v I. l v spectrum 0 t e actmty lSC osed in t is invention. (Im/a mrinm rll'z'lmidz'x 6.0 3.0 0 Smee no firm rules of procedure can be la d down for Sig berm. 1 O 1 x U the sequence of such evaluations or for the choice of pathogens, diethyl alpha allyl phosphonate must be 'j l 'l' -"l considered on the basis of its demonstrated perfor- Ammum, 33 L8 0 rnancem such primar v evaluations and then progres- ATCC No. 639 z sn'ely udged in subsequent studies. A wide range of n g U pathogens. representative of economically important l'crririllimn aIIm-ulrum 3.x 2 3 1.0 diseases. can be used to help define diethyl alpha allyl phosphonates biological activity and to asssure high degrees of success under field conditions. The following disease organisms, crops and reference standards may be used in such evaluations:

Reference Disease Disease Organism Compound Powdery Mildew of Eryxiphc ciclmmceu- Maneb. Cucumbers rum Karathane Leaf Rust of Wheat Pucciniu rubigu-i'tm Maneb.

Karathane Leaf Rust of Wheat Put't'inia ruhigo-rm'u Plant ax Rice Blast Disease Piriculuriu O)' \I,tl(' Blasticidin Downey Mildew of Sugar Pemnusporu .rv/mcrii Karathane Beet Downy Mildew of Lima Plrvmphrlmm plmxe- Karathane Bean uli Bean Rust L'mmyces plmxmli Maneb \"ur. Iypitu Powdery Mildew of Wheat Erysip/w graminis Karathane Powdery Mildew of Apple PmInxp/mcru leucu- Karathane Iric/m Powdery Mildew of Roses .S'plmvrurlu'm pmumm Karathane var. msuc Powdery Mildew of Erywiplw t'it'hm'ut'ew Karathane Cantalope rum Leaf Spot of Wheat Hvlmi:uImx mrium Maneb .mlirnm Early Blight of Tomato Allw'mu'iu .mlum' V Maneb Rice Blast Disease Piriculuriu urygm Blasticidin C erocspora Leaf Spot of (en'uxpura Imtitulu Maneh Sugar Beets Septoria Leaf Spot of Sepmriu upii- Maneh ,iuureulmllix Celery Apple Scab Venluriu mum unlit C yprex Common Bacterial Blight X uni/murmurs [)lklSt'O Streptoof Bean mycin Sulfate Wherever possible, the applicants recommend the use of in vivo procedures to test the diethyl alpha allyl phosphonate compositions of this invention to demonstrate their efficacy under more realistic conditions. However, not all pathogens lend themselves to such techniques. In order to provide additional spectrum definitions, the following fruit-rotting, storage decay and bacterial pathogens may be tested by in vitro methods: 1

Brown Rot of Stone .S'clerminiu j'rucliomlu C aptan Fruits Grey Mold of Fruit and BOIl' \iS ('im'reu Maneb Vegetables Rhizopus Fruit and Rlzl'wpus nigrit'anx Maneb Vegetable Rot Citrus Green Mold Penicillium digimlum Maneb Citrus Blue Mold Pelvic-[Ilium imlicum Karathane Bacterial Disease of Pseuzlmnomm' stringue Captan Many Fruit Crops Bacterial Soft Rot Erwinia cammruru Captan In their plant protection aspects, the diethyl alpha allyl phosphonate compounds of this invention may be used in the manner known to the organophosphorus crop protection art; that is, they can be made up in solid or liquid formulation. Examples of solid formulations are dust, wettable powders, granules and pellets. As a dust, diethyl alpha allyl phosphonate compounds may be dispersed in powdered solid carriers such as talc, soaps, soapstone, attapulgus clay as well as other finely divided solids known to the dusting art. When formulated as wettable powders, the active component may be employed in conjunction with inert fillers which may be of the clay type carrier or non-clay type, in conjunction with various combinations of wetting agents and emulsifiers which permit the adaptation of the con- 10 centration as a free-flowing powder for dispersion in the field.

Each of these carriers may in turn contain other carriers or extenders which are ordinarily non-reacting or inert substances such as sand, clays, talc, sawdust, alkaline earth carbonates, oxides, phosphates and the like as well as diatomaceous earth, micas or other suitable materials. When liquid formulations are desired, liquid extenders, dilutants or carriers of a nonreactive nature may be utilized, Examples of such materials are alcohols, ketones, glycols, aromatic hydrocarbons, petroleum fractions such as octane and various other distillates. From these considerations, it will also be recognized that the above formulations with slight modifications may be used in the field of animal husbandry as dusting powders and salves.

Where it is desired to use the aforementioned wettable powders or liquid formulations, either emulsified, dispersed or suspended in water or other fluids, one or more of the class of materials herein referred to as adjuvants can also be incorporated into the powder, dust or liquid formulation. These adjuvants comprise surface active agents, detergents, wettable agents, stabilizers, dispersing agents, suspending agents, emulsifying agents, spreaders, stickers and conditioning agents generally. To their modifying characteristics these adjuvants may facilitate handling and application and infrequently enhance or potentiate the diethyl alpha allyl phosphonate compositions of this invention in their biological activities by mechanisms which are frequently not well understood. A satisfactory but not exhaustive list of these adjuvants appears in Soap Chemical Specialties, Volume 31, No. 7, Page 61; No. 8, Pages 38-61; No. 9, Pages 52-67; and No. 10, Pages 3867 1955 See also, Bulletin No. 697 of the Bureau of Entomology and Plant Quarantine of the United States Department of Agriculture.

An additional advantage of diethyl allyl phosphonate is its compatibility with a variety of other biocidal and fungicidal materials. For example, it may be convenient to combine diethyl alpha allyll phosphonate with one or more other adjuvants, carriers, pesticides, biocides or fungicides of various structures. For example, diethyl alpha ally] phosphonate fungicidal inhibitors may be combined with insecticidal materials such as chlordane, benzene hexachlorides, DDT, DDD, the insecticidal carbamates, polychlorinated terpenes, parathions, methozychlor, insecticidal phosphates, phosphorothioates, phosphorodithioates and with fungicides such as sulphur, quinones, dodecylgaunidine and metal dimethyldithiocarbamates.

There are many other considerations such as concentration and method of application which may make some methods of application more favored than others. These considerations may include the type of organisms on which the compound. is to be administered, the degree of activity, the degree of activity toward the particular organism, and side effects. Also to be considered is the cost of production and the characteristic solubility of diethyl alpha allyl phosphonate in the carrier material.

The broad spectrum of antifungal activity afforded by diethyl alpha phosphonate can also be utilized in the formulation of disinfectant solutions, paints, coatings. films and polymeric materials in order to protect against disease and rot caused by various fungi species. When used as a disinfectant, suitable formulations may be prepared by mixing the compound with an emulsifying agent in the presence of organic solvents and then diluting it with water to form an aqueous emulsion containing the diethyl alpha allyl phosphonate. Suitable emulsifying agents include. e.g. alkylbenzenesulfonates, polyalkene glycols, et cetera. Aqueous emulsions of diethyl alpha allyl phosphonate are particularly suited for use in disinfectant solutions used in washing hospital floors and walls. The following examples will further illustrate the antifungal properties of diethyl alpha allyl phosphonate.

EXAMPLE Vlll PREPARATION OF A VINYL COATING Resistant to Mildew Deterioration A vinyl coating is prepared using a commercially available preparation without a fungal growth inhibitor.

An identical vinyl coating was prepared except that 2% by weight of diethyl alpha allyl phosphonate was incorporated into the coating formulation.

Two sets of components such as asbestos tubing, silkwrapped transformers and rayon-wrapped solenoids were obtained. One set was sprayed with the vinyl coating containing inhibitor, the other with the identical coating without inhibitor.

EXAMPLE lX PREPARATION OF PLASTlClZERS Resistant to Mildew A commercial thermoplastic monomer was divided into portions which were treated as follows.

Portion 1: To the first portion was added 2% by weight of diethyl alpha allyl phosphonate and by weight of dimentylnaphthalate as plasticizer. The monomer is polymerized and molded into 3inch diameter discs, inch in thickness prior to testing.

Portion 2: To this portion was added 2% by weight of diethyl alpha allyl phosphonate and 10% by weight of butyl isodecylphthalate as plasticizer. The monomer is polymerized and molded as above.

Portion 3: This portion was the untreated control of Portion 1 containing no fungal inhibitor but 10% by weight of dimethylphthalate as plasticizer. Again, the polymerization and molding are identical.

Portion 4: This portion was the untreated control of Portion 2 containing no fungal inhibitor but 10% by weight of butyl isodecylphthalate as plasticizer. The polymerization and molding are as described above.

The two plasticizers were chosen on the basis of their known susceptibility to F Usarium attack under high humidity and temperature conditions.

EXAMPLE X VINYL COATINGS AND PLASTICIZERS For Fungal Resistance The vinyl coated articles and controls of Examples V111 and 1X were placed in an air-tight high temperature and high humidity chamber maintained at 80 F. and 95% humidity to simulate tropical temperature and humidity conditions. After a months exposure the vinyl coated articles treated with inhibitor were only slightly attacked by rot while the articles coated with vinyl without inhibitor were well rotted. The two untreated control polymer discs are examined and were found to be blackened and mildew rotted. Isolates of Aspergillus, Fusarium and known species of yeasts were prepared from the deteriorated discs. The discs 12 containing the diethyl alpha-allyl phosphonate fungal inhibitor were not adversely affected.

EXAMPLE X1 ALGAECIDAL PROPERTIES OF DlETl-lYL ALPHA ALLYL PHOSPHONATE The effectiveness of diethyl alpha allyl phosphonate against various algae species was established in the following manner. Algal cultures representing Scenedesmus, Chlorella, Plectonema, Anacystis, Ankistrodesmus, Anabaena, Synura, Oscillatoria, Coccochloris, Chlamydomonas and Lyngbya were each maintained in Chu No. 10 Broth Medium (Calcium nitrate, 0.040 grams; Potassium phosphate, 0.010 grams; Magnesium sulphate, 0.025 grams; Sodium carbonate, 0.020 grams; Sodium silicate, 0.025 grams; Ferric citrate, 0.003 grams; Citric acid, 0.003 grams; and deionized water; 1.000 ml.) in the presence of sunlight. Hardened Chu No. 10 agar plates were inoculated with cotton swabs saturated with the respective algae broth cultures. The diethyl alpha allyl phosphonate was tested by impregnating filter paper discs (1.27 cm. in diameter. No. 740-E, Schleicher and Schuell, Keene, New Hampshire) with a solution of the diethyl alpha allyl phosphonate at the concentrations indicated. The saturated filter discs were placed on the surface of the seeded agar plates and the optimum growth temperature of 25 to 27 C. was maintained. the results of these tests are expressed as inhibition zone diameters.

Inhibition lnhibition Inhibition Zone (cm) Zone (cm) Zone (cm) for for for Algae ug/disc l0 ug/disc 3 ug/disc Scmwdemnus Imxil- 1.7 .75 0

crisis Taft EEC 83 Sccnerlrsnrux ubli- 10h 10h 0 quus Taft EEC 92 Svenvdawnu; ohli- 4.7 6.4 0 quux SR1 EEC 315 Chlorella \ulguris tr 0 0 ATCC No. 9765 Pleclmwmu mmuum 2.5 1.8 1.0 Taft EEC 172 Almqxtlllt nizluluns tr 0 O Taft EEC 134 .-lnki.r1rmlamzux T0 1 1 0 NH. miculm'is Taft EEC 28 Anubuenu culenulu 4.7 4.0 2.5 SR1 EEC 315 S \'uuru sp. 5.0 5.0 0 U1 ()xcillulul'iu cur- 1.8 1.0 0 Mali Ul Cucc'oclzluris zle- 1 7 0 0 Imus SRl (Ir/mnydmnulms Furl!" 1.8 1.0 0 (Hi UA L vy/ r11 sp. 1.9 0 0 Taft EEC 166 Comments: h hazy zone tr trace As was the case with the bacteria, it should be recognized that the scope of this invention should not be limited to any particular species. For instance, the algaecidal activity of diethyl alpha allyl phosphonate against Chlorella vulgaris suggests that the compound will also prove to be of value against such other Chlorella species such as Chlorella ellipsoidea, C lzlorella pyrenoidusa. C lrlorella variegam et c etera. Similar possibilities exist for species of other genera whose activity was shown to be arrested by diethyl alpha allyl phosphonate. It should be recognized also that other appropriate algae genera may well afford additional opportunities to further define the degree and spectrum of the algaecidal activity disclosed in this invention. Since no firm procedure can be laid down for the sequencing of such evaluations or for a selection among the more than 20,000 known algae species, the diethyl alpha allyl phosphonate compounds of this invention must be considered on the basis of their demonstrated performance in these primary evaluations and then progressively judged in subsequent studies. These evaluations should include but not be limited to the following algae gen era.

TASTE AND ODOR CAUSlNG ALGAE GENERA Rlziwclonium Merisnmpmliu Meridian Pimmluriu Ap/umulluw Cln'mlmlinu L'lurlirix Plum-mus Clmluphuru Rhmlnnmrmx Am'iz'ulu .Smm'ustrum Surircllu C/mnulesiplmn Le/mmeu Cyclolella Mir'rnxlm'im' Cmcuncix Chr \'.ru('0cm.s' Culur/zrix M forum/Bus Ankislrmlcsmux FILTER CLOGGING ALGAE GENERA Annbmnu ('lusm'ilml Spimgyru C/UUUL'IML'HS Tubelluriu 'I'ruc/ielunmnux Dinnhryun Rirulariu Asreriomllu Cyril/1011a Meloxiru Palmella Chlurcllu clulellu Diummu Synezlra Auriculu I-rugilurai Trihunemu ()xt'illumriu POLLUTED WATER ALGAE GENERA A l'rllrospiru Terran/r011 .4 nubuenu Merixnmpediu Euglyuu P/lar'us P/mrmidimn Spirog 'ru Glueognpsu Curleria Clllurucut'cum Sligmclnninm Lepucim'lix ()sillumriu Gumplzum'nm A'ilzxclzia L \'ng/2yu Chlamyzlomunux Chlumyduhmryx SURFACE WATER ALGAE GENERA The scope of this invention should encompass the use of this compound in waters of all types such as lakes, rivers, ponds, streams, reservoirs, swimming pools and oceans as well as recirculating industrial waters. These compounds can be used to prevent the initial occur- 14 and most fish food organisms at algae killing concentrations.

Another important advantage of diethyl alpha allyl phosphonate compounds in their algaecidal applications is that they can be made up in solid or liquid formulations. Examples of solid formulations are dust, wettable powders, granules and pellets. Solid formulations, particularly floating solid formulations. may be preferred in combating algae which grow on surface waters. As a dust, diethyl alpha allyl phosphonate compounds may be dispersed in powdered solid carriers such as talc, soap, soapstone, attapulgus clay, as well as other finely divided solids. When formulated as wettable powders, the active diethyl alpha allyl phosphonate component may be employed. in conjunction with inert fillers which may be of the clay type carrier or non-clay type in conjunction with various combinations of wetting agents and emulsifiers which permit adaptation as a free flowing powder. Each of these carriers may in turn be combined with other carriers which are ordinarily nonreacting or inert substances such as sand, clays, talc, sawdust, alkaline earth carbonates, oxides, phosphates and the like, as well as diatomaceous earth, micas or other suitable materials.

When liquid formulations are desirable, liquid extenders or carriers of a non-reactive nature may be utilized. These compositions should contain approximately 0.1 to 20% by weight and preferably 0.1 to 3% and most preferably 0.5 to 2% of the active diethyl alpha allyl phosphonate ingredient. Solvents which may be used in the preparation of such compositions would include alcohols, ketones, glycols, mineral spirits and aromatic solvents such as benzene, xylene, nitrobenzene, dimethylformide. Furthermore, to assist in the rapid and complete dispersion in water systems, these compositions may also contain approximately 5 to 30% by weight and preferably 10 to 15% by weight of surface-active agents. Suitable surfaceactive agents include sodium dialkyl; sulphates, sodium alkylbenzene sulfonates, sodium carboxylates and the non-ionic surfactants such as ethoxylated. fatty acid alcohols and amines.

Diethyl alpha allyl phosphonate is compatible with a wide variety of other algaecidal, biocidal and fungicidal materials. For example, it may be convenient to combine one or more diethyl alpha allyl phosphonate compositions with one or more of the other biocides, fungicides or algaecides. For example, common fungicides and biocides such as sulphur, inorganic salts such as copper sulphate, activated colloidal silver compounds, copper naphthenate and zinc acetate, as well as substituted hydrocarbons and quarternary ammonium compounds, may be employed.

Considerations concerning concentrations and method of applications may make some methods of application and use of diethyl alpha allyl phosphonate more favored than others. These considerations may include the type of organisms on which the compound is to be administered, the degree of activity, the degree of inhibition toward the algae organism and possible side effects. Also to be considered is the cost of production and the characteristic solubility of diethyl alpha allyl phosphonate in the carrier compounds.

In their algaecidal aspects the applicants have discovered that diethyl alpha allyl phosphonate compounds are active algaecides at relatively low concentrations. For example, it has been discovered that these compounds have imparted algaecidal activity at concentrations as low as 0.1 ppm. The amount of diethyl alpha allyl phosphonate added to the water will, of course, vary depending upon such factors as the tyupe of algae present, the nature of the body of water, i.e. flowing stream versus small lake, et cetera, and the inherent ability of the body of water to support algae growth. This inherent ability in turn depends upon such factors as exposure to sunlight, pH, nutrient capabilities, and the like. In most cases, however, the concentration of diethyl alpha allyl phosphonate required to kill or inhibit growth of algaes will vary from 0.1 to 10 ppm, the preferred amount is in the range of 0.8 to 5 ppm. These compounds can be added to the water according to conventional techniques for algaecide applications. When treating a lake or body of water which is relatively calm, the conventional procedure is to spray as aqueous solution of the algaecide over the surface of the water. The diethyl alpha allyl phosphonate generally will be predissolved in the types of solvents previously mentioned. In the case of moving water, such as in a water treatment plant or industrial facility, the algaecide can be added to the water in small amounts at periodic intervals. For economic reasons, volume usages such as in lakes, streams, reservoirs, as distinguished from specialized uses such as in aquatic gardens, and industrial applications, the concentrations of the diethyl alpha allyl phosphonate algaecides probably will not be more than 0.8 to 5 ppm of the water containing the algae.

Having thus disclosed our invention, we claim:

1. A method of killing and inhibiting the growth of algae which comprises contacting said algae with diethyl alpha allyl phosphonate in an amount effective to kill and inhibit the growth of said algae.

2. A method of claim 1 wherein the algae are contacted with growth inhibiting amounts of diethyl alpha allyl phosphonate together with acceptable carriers in an amount effective to kill and inhibit the growth of said algae on a plant infected with said algae.

3. The method of claim 1 wherein the algae is Chlorella vulgaris.

4. The method of claim 1 wherein the algae is Plectonema notatum.

5. The method of claim 1 wherein the algae is Ankistrodesmus acicularis.

6. The method of claim 1 wherein the algae is Anabaena calenula.

7. The method of claim 1 wherein the algae is Synura U1.

8. The method of claim 1 wherein the algae is Oscillatoria comeli.

9. The method of claim 1 wherein the algae is Coat-0- c/zloris elebans.

10. The method of claim 1 wherein the algae is Scenedesmus basilensis.

11. The method of claim 1 wherein the algae is Scenedesmus obliquus. 

1. A METHOD OF KILLING AND INHIBITING THE GROWTH OF ALGAE WHICH COMPRISEES CONTACTING SAID ALGAE WITH DIETHYL ALPHA ALLYL PHOSPHONATE IN AN AMOUNT EFFECTIVE TO KILL AND INHIBIT THE GROWTH OF SAID ALGAE.
 2. A method of claim 1 wherein the algae are contacted with growth inhibiting amounts of diethyl alpha allyl phosphonate together with acceptable carriers in an amount effective to kill and inhibit the growth of said algae on a plant infected with said algae.
 3. The method of claim 1 wherein the algae is Chlorella vulgaris.
 4. The method of claim 1 wherein the algae is Plectonema notatum.
 5. The method of claim 1 wherein the algae is Ankistrodesmus acicularis.
 6. The method of claim 1 wherein the algae is Anabaena catenula.
 7. The method of claim 1 wherein the algae is Synura UI.
 8. The method of claim 1 wherein the algae is Oscillatoria corneti.
 9. The method of claim 1 wherein the algae is Coccochloris elebans.
 10. The method of claim 1 wherein the algae is Scenedesmus basilensis.
 11. The method of claim 1 wherein the algae is Scenedesmus obliquus. 